Hirudin-PA and its derivatives, process for manufacturing these and the use of these substances

ABSTRACT

The present invention relates to a new content substance found in leeches (Hirudo medicinalis), the derivatives of this abbreviated at the N-terminus and the C-terminus, and the desulfated derivatives therefrom as well as a process for the production of these compounds, and their use. These compounds possess thrombin-inhibiting properties and can be used to inhibit clotting in human and animals, for blood conservation, or as reagents for the analytical determination of thrombin.

FIELD OF THE INVENTION

The present invention relates to hirudin-PA and its derivatives, aprocess for manufacturing these substances, pharmaceuticals that containthese substances, and the use of these substances.

BACKGROUND OF THE INVENTION

Several substances have already been extracted from medicinal leeches(hirudo medicinalis) such as polypeptides that act as proteinaseinhibitors and which have, in part, an antithrombin action. In theliterature that deals with these substances, a distinction is drawnbetween the so-called eglines and hirudins. Two eglines are described inDE-PS No. 28 08 396. The extraction of crude hirudin is described in DiePharmazie, Number 36, 1981, pages 653-660 and in Methods in Enzymology,Volume 45, 1976, pages 669-678. The complete amino acid sequence isknown from FEBS 1104 (Federation of European Biochemical Societies,Volume 165, 1984, pages 180-184).

SUMMARY OF THE INVENTION

Most surprisingly, it has now been found that crude hirudin containsanother component that is pharmacologically active.

For this reason, the present invention undertakes the examination ofleech extracts for new pharmacologically effective substances, inparticular the hirudin components of leech extracts more precisely fornew substances.

According to the present invention, the solution to this task lies inthe preparation of new hirudin components, designated hirudin-PA, theirdecomposition products, and the desulfated derivatives therefrom.

The present invention relates to hirudin-PA of formula I: ##STR1## thederivatives of these substances, shortened at the N-terminus by up to 2amino acids and at the C-terminus by up to 17 amino acids as well as thedesulfated derivatives, in which, in the above formula I: (i) thesulfate ester group at the phenolic hyroxyl of the tyrosin group inposition 64 is missing, or (ii) the sulfate ester group described at (i)and the latter or the two last amino acids D, E (in position 66 or66+65) are missing, and the pharmaceutically useful salts thereof.

A preferred embodiment are hirudin-PA and its derivatives of formula I,the amino acid chain at the N-terminus being shortened by the sequence Ior IT.

A further preferred embodiment are hirudin-PA and its derivativeswherein the amino acid chain at the C-terminus is shortened by thesequence: ##STR2##

Especially preferred is hirudin-PA of the formula I ##STR3## and itssulfated derivatives as defined above.

In the preceding formulae the quoted letters symbolize the proteinogenicamino acids that are peptidically linked, these letters corresponding tothe IUPAC nomenclature. The salts of these proteins are also the objectof the present invention.

The hirudin-PA according to the present invention consists of a total of66 amino acids; its molecular weight is 7087; its specific antithrombinactivity is 680-720 IU/mg and the complex with thrombin has thedissociations constant Ki=4×10⁻¹¹ M.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the yields of PTH amino acids of the oxidizedhirudin-PA;

FIG. 2 is a graph showing the yields of PTH amino acids of trypticpeptide TR 16;

FIG. 3 is a time diagram for the CPY catalyzed liberation of the aminoacids of the C-terminus of hirudin-PA; and

FIG. 4 shows the complete sequence for hirudin-PA.

DETAILED DESCRIPTION OF THE INVENTION

Hirudine-PA and its derivatives are structurally very similar to thealready known hirudin. However, at several important places on theprotein chain, particularly at the beginning and at the end, theproducts according to the present invention differ in a characteristicmanner from the sequence of the known hirudin. This results in furtherdifferences that are of great practical significance. The tertiarystructure of these polypeptides is stabilized by three disulfidebridges. In hirudin-PA the strongly acid sulfate monoester group on thephenolic hydroxyl of the tyrosin group in position 64 is conspicuous;this can be represented by the following partial-structure formula:##STR4##

This grouping is also present in the known hirudin, although in this itis in position 63.

From the biological standpoint, hirudin with a Ki value of approximately3×10⁻¹¹ M belongs amongst the most effective thrombin inhibitors; itacts completely specifically against thrombin and inhibits no otherproteinases of the blood clotting cascade. Unlike heparin, the hirudinsaccording to the present invention exert their inhibiting influence onthrombin directly. Of their pharmacological effects, the inhibition ofblood coagulation is important. The substances are thus particularlywell suited for the prophylaxis of thromboses. No undesirableside-effects have been noted up to the present. During intravenousadministration to dogs and human subjects there was no effect on heartrate, respiration, blood pressure, thrombocyte count, fibrinogens andhaemoglobin. In comparative tests the compounds according to the presentinvention were superior to heparin.

In order to produce the hirudins according to the present invention oneproceeds best from the known leech extracts. As has been discussedabove, the production of extracts of this kind is described, forexample, in Die Pharmazie, Number 36, 1981, pages 653-660 or in Methodsin Enzymology, Volume 45, 1976, pages 669-678.

One proceeds in that leeches (hirudo medicinalis) or the head ends ofleeches are reduced, homogenized, the tissue paste extracted withaqueous acetone or salt- or buffer-solution, a significant proportion ofthe impurities of the extract precipitated with ethanol duringfractionation, the solution is reduced in a vacuum, the reduced solutionis subjected to fractionating acetone precipitation, the precipitateobtained at higher acetone concentrations is reduced, this then beingextracted, and the extract lyophilized. Proceeding from thislyophilisate that is familiar in and of itself, the process according tothe present invention is characterized in that: (A) the lyophilisatethat is obtained is chromatographed on a Sephadex column that has beenequilibrated with a buffer of pH 7.8; (B) the fractions withantithrombin activity are lyophilised, the lyophilisate is desalinated,and lyophilised once again; (C) an anion exchange chromatography iscarried out on DEAC cellulose with the lyophilisate, equilibration beingcarried out with a buffer of pH 6.5 and elutration being carried outwith a buffer of pH 6.0; (D) the fractions that are antithrombin activeare lyophilised, desalinated, and lyophilised; (E) the lyophilisate isplaced in a DEAE-Sephadex column that has been equilibrated with abuffer of pH 6.0, elutriated with a buffer solution with a linear pHgradient that is formed from a buffer of pH 5.0 and a buffer of pH 3.7;(F) the fractions that are antithrombin active are elutriated atapproximately pH 4.6 to 4.7 are lyophilised, desalinated, and againlyophilised; (G) finally, the lyophilisate is chromatographised on anHPLC column that is filled with a reversed phase sequestering agent oftype C₁₈, with 0.1% trifluoracetic acid in water serving as elutriant(A) and 0.1% trifluoracetic acid in acetonitrile with 40% (A) (v/v)serving as elutriant (B)), with isocratic conditions ((63% (A)+37% (B))predominating, when one obtains four thrombin-suppressing fractions,with the first of these containing the desired hirudin-PA; (H) toproduce the desulfated forms of the hirudin-PA the sulfate monoestergroup on the phenolic hyroxyl of the tyrosin group in position 64, andif desired the amino acids D, E standing at the ends in positions 66, 65are split off hydrolitically, either singly or together; (I) in order toproduce the reduction decomposition products of the hirudin-PA this isincubated with a peptidase, the products separated through RP-HPLC, andthe individual products isolated and lyophilised.

It is preferred that the chromatography in stage A be carried out onSephadex G75, with an aqueous solution that contains 50 mMtriethanolamin and 300 mM or 400 mM NaCl is used as a buffer of pH 7.8.In addition 0.02% NaN₃ can be added to this and to the buffer solutionsdescribed below.

It is preferred that the anion exchange chromatography be carried out onWhatman DE52 or DE53, with an ammonium acetate buffer (30 mM NH₄ Ac andoptionally 0.02% NaN₃) used as an equilibrating buffer and a sodiumacetate buffer composed of 0.2M NaAc and 0.19M NaCl used as anelutriation buffer with pH 6.0. It is also possible to use a saltgradient as an elutriation buffer, when one works first with 0.03Mammonium acetate, 0.19 m NaCl, pH 6.5, and finally with 0.03M ammoniumacetate, 0.3M NaCl, ph 6.5.

It is preferred that a sodium acetate buffer of the above composition beused as a buffer of pH 6.0 during the Sephadex DEAE-A25 chromatography.The same buffer, on the one side at pH 5.0 and on the other at pH 3.7,is used to produce the pH gradient. The fractions that are elutriated atapproximately pH 4.6 to 4.7 are antithrombin active and are designatedhirudin pool 1. Fractions that are elutriated at approximately pH 4.4are isolated as hirudin pool 2. The pool 1 that contains theantithrombin activity is then lyophilised, desalinated, and lyophilisedagain.

The desalination operations are as a rule carried out with Sephadex G25,with water serving as the wash agent. For the HPCL separation one canuse, for example, a Type LC-18-DB Supelco column 5μ or a Type 100CH-18/2 Lichrospher column, 5μ4.6×250 ml, by Merck.

Using a column of this kind, one works at a flow of 1 ml/min, at atemperature of 25° C., and detects at 214 and 254 nm.

During this HPLC separation of the hirudin pool 1 one obtains forthrombin-inhibiting fractions. The largest portion is identical with thehirudin known from the literature. Another small fraction differs fromthe known hirudin only by the exchange of an amino acid. The hirudin-PAaccording to the present invention is contained in the first and secondstrongest fraction. It is a completely new inhibitor type withN-terminal Ile and is described and sequenced as follows:

The protein of the above-described hirudin-PA fraction was immobilisedin a solid phase and in this state subjected to the so-called Edmandecomposition process. The standard technique of automatic solid phasesequencing employed in this instance is described, for example, in twosurvey articles by W. Machleidt Modern Methods in ProteinChemistry--Revue Articles, published in 1983 by Walter de Gruyter & Co.,Berlin, New York, and by Richard A. Larsen and W. Machleidt in Methodsof Biochemical Analysis, Volume 26, pages 201 to 284, 1980. The peptidethat is to be sequenced is bonded covalently to the insoluble carrier,the solid phase, through functional groups of the amino acid sidechains. In the solid phase it passes through the cyclic reactions of theEdman decomposition that lead to the separation of the specificN-terminal amino acids. The bonding of the peptide to the solid phasemust be stable under the conditions of the Edman decomposition but mustnot, however, hinder the decomposition up to the C-terminus.

During this procedure, the phenylthiourea derivative of the cysteinicacid is particularly well-suited for the positive identification of theposition of the cystine group. For this reason, sequence runs withnative and oxidized hirudin-PA are carried out to clarify the N-terminalsequence. 25 nMol protein is used per run, half being immobilised thoughamino groups on diisothiocyanate glass (DTIC glass), and half throughcarbodiimide coupling of the carboxyl group on aminopropyl glass (APG).These two immobilizing techniques are described, for example, on pages270 and 273 of the above references in W. Machleidt Modern Methods inProtein Chemistry, 1983. Since lysin groups couple at a high yield toDITC glass through their α-amino groups, mixed coupling entails theadvantage that both the N-terminal amino acids and the position of thelysin radical can be determined in one sequence run.

It was first possible to clarify the sequence of the hirudin-PA up toposition 46 (FIG. 1). The positions of the cystein groups and the lysingroups were established positively, although their phenylthiourea (PTH)derivatives were not quantified. Open circles symbolise the PTHderivatives of Asp, Glu, Ser, Thr and Pro. They represent those aminoacids, of which the yields of PTH derivatives was lower. The repetitiveyields of the sequence run of oxidized hirudin-PA (FIG. 1) wascalculated as 94% for steps 10 to 34. At position 35, the lys 35 anchorpoint, the yield of PTH amino acids drops abruptly. These data indicatethat still only a little material was bound through the last aminogroups - anchor point 47, and at the same time the yields for thecarboxyl groups coupling were low.

FIG. 1. Yields of PTH amino acids of the oxidized hirudin- PA.

The solid circles in positions 10 to 34 are used to calculate theregression lines. A repetitive yield of 94% resulted from the slope. Theopen circles were used for Asp, Glu, Pro, Thr, and Ser, which are eitherpartially fixed to the carrier through side chains (Asp, Glu) orincompletely separated (Pro), or partially destroyed during thedecomposition (Thr, Ser). Lysin was identified positively, but notquantified, however. Tryptic decomposition of oxidized hirudin-PA.

19 protein peaks were obtained during the separation of a trypticseparation batch by means of RP-HPLC. Description of the peptides wascarried out by amino acid analysis and N-terminal determination with aone-step Handedman decomposition (DABITC/PITC double-coupling method)(Chang, Brauer, Wittmann-Liebold, 1978, FEBS-Lett., Number 93, pages 205to 214.). The TR 3 peptide was identified as tripeptide G-N-K that wasassociated with the known positions 25-27. The TR 16 peptide containedthe C-terminal peptide with the former overlapping sequence information,beginning with 36 (Asp). After coupling to APG this was subjected tosolid-phase Edman decomposition. The yield of these PTH derivatives wasonce again significantly lower, this being caused by the fixation of Aspand Glu. What has been said above also applies to the other derivatives.The yield of the second Edman step (Asn) was set at 100% and the otheryields related to this figure. The slope of the regression lines for thesolid circles revealed a repetitive yield of 93.3% (FIG. 2).

Comparison of the amino acid analysis and the amino acid compositionafter sequencing revealed a discrepancy of one amino acid:one Glx tooestablished within the sequence. This amino acid had to the C-terminus,otherwise no indication of an additional Glx have been noted. AC-terminus sequencing with carboxypeptidase Y should be completed so asto clarify the C-terminus sequence and answer the question as to whetherthe tyrosin group is a tyrosin-O-sulfate groups as in the hirudin.

FIG. 2: Yields of PTH amino acids of tryptic peptide TR 16.

The yield from the second decomposition step according to Edman has beenset at 100% and the remaining yields related to this. The solid circleswere used to calculate the regression lines, and a repetitive yield of93.3% was obtained from their slope. Open circles were used for Asp,Glu, Pro, Thr, and Ser amino acids (See FIG. 1). C-terminal sequencingof hirudin-PA with carboxy-peptidase Y (CPY).

The C-terminal sequencing of hirudin-PA is carried out from nativehirudin-PA, the TR 16 peptide, and the TR 16 peptide that had beenincubated previously for 30 minutes at 60° C. with 50% TFA. Initially,rapid separation of glutamin acid was observed prior to liberation ofaspartic acid and additional amino acids (FIG. 3). The liberation oftyrosin was followed only in the TR 16 peptide that had been treatedwith TFA, whereas the effect of CPY on the two other samples led toseparation of an amino acid that behaved in the same manner as synthetictyrosin-O-sulfate. Thus, the tyrosin group in position 64 of thehirudin-PA as also identified as a tyrosin-O-sulfate group. However, themost surprising result was determination of Glu as a C-terminal aminoacid. Since no attachment point for this group had been ootained duringthe automatic sequencing of the TR 16, it had to have been coupled tothe carrier completely through C-terminal and side chain carboxylgroups, which meant that it was not accessible to an identification. Aspis only poorly hydrolysed by CPY and thus constitutes a limiting factorin the decomposition. It is thus rendered more difficult to coordinatethe series of the C-terminal amino acids, since two Asp are presentclose to each other in the sequence, and Pro had not been detected. Forthis reason, the C-terminal sequence by CPY decomposition should only beindicated as -Tyr-Asp-Glu.

FIG. 3: Time diagram for the CPY catalyzed liberation of the amino acidsof the C-terminus of hirudin-PA.

Hirudin-PA was incubated with CPY (40/1=w/w) in a 10 mM phosphate bufferpH 4.7 at 37° C. Aliquotic samples were taken from the incubation batchat specific times, adjusted to pH 2.0 and lyophilised. The lyophilisatewas analyzed for liberated amino acids on the amino acid analyzer. Noprolin was detected. The C-terminal sequence was determined as-Tyr-Asp-Glu.

The complete sequence for hirudin-PA is reproduced below and is shown inFIG. 4. ##STR5## Sequence of the amino acid blocks in hiruding-PA,identified as follows: ←→direct Edman decomposition of native andoxidized hirudin-PA

←-→Edman decomposition of trytic peptide

←←←enzymatic decomposition by CPY

The decomposition products of the hirudin-PA according to the presentinvention can be obtained by enzymatic decomposition of the hirudin-PAin the form of a limited proteolysis. To this end, one uses peptidases,such as carboxydiases, i.e., proteases, which split off an amino acidchain from the carboxyl end and/or amino peptidases, i.e., proteasesthat attack an amino acid chain from the amino end. Carboxypeptidases A,leucinaminopeptidase and the A,B,C, and D kathepsines are suitableproteases that can also be bound to the carrier; however,carboxypeptidase Y and kathepsin C are particularly well suited.

It is known that kathepsin C as a dipeptidyl-aminopeptidase splits offdipeptides sequentially from the unsubstituted amino end of the protein.However, it was found that kathepsin C also has C-terminal exopeptidaseactivity during the proteolysis of hirudin, so that the decomposition ofhirudin-PA with kathepsin C can also be managed from the C-terminal end.

It is preferred that the proteolysis of hirudin-PA be done withkathepsin C. Incubation is best carried out at 37° C. The pH optimum ofthe enzymatic activity of kathepsin C lies in the weakly acidic area ofDH 5 to 6.

The separation of the product mixture obtained after proteolysis isconducted with HPLC. As an example, one can use a Supelco columnLC-18-DB or a Lichrospher column 100 CH-18/2. A gradient of 0.1%trifluoracetic acid in water (V/V; Buffer A) and 0.1% trifluoraceticacid in acetonitrile with 40% bufferA (V/V)has proved to be effective asan eluting agent.

According to the present invention the desulfohirudins-PA can beobtained if one liberates the phenolic hydroxy group of the tyrosinradical in position 64, present as sulfuric acid monoester in hirudin-PAof the formula given above.

Liberation of this group corresponding to the formula

    HO--SO.sub.2 --O--pept  HO--pept

(wherein pept stands for the residual portion of the hirudin) can becompleted, for example, by hydrolysis, both chemical and biologicalmethods being applicable for this purpose.

During chemical liberation it is preferred that the process be completedunder the general conditions of acid catalysed hydrolysis, as under theaction of a diluted aqueous hydrochloric acid solution, e.g., containing2- to approximately 4N, preferably in trifluoracetic acid, as reactionmedium, or with trifluoracetic acid that contains water, alone asreaction agent and solvent. In order to keep the danger of thehydrolitic separation of peptide compounds to a minimum, it isrecommended that work be carried on under mild reaction conditions,e.g., at temperatures not in excess of room temperature, and theprogress of the reaction be monitored analytically, i.e, by means ofthin-film chromatography.

However, the hydrolysis is best completed by biological methods, inparticular by the use of specific enzymes, arylsulfatases, which splitoff the phenolic sulfate ester groups to free phenolic groups under mildconditions. The biological liberation of the sulfated hydroxyl groupscan be completed with the help of a suitable enzyme preparation with anenriched substance biocatalyst or an isolated enzyme, or one can use asuitable enzyme system in situ, as is immediately available, e.g., agrowing or static microorganism, a cell culture, a cell homogenate, oran autolysate. One of the greatest advantages of biological hydrolysisis its high level of selectivity, that brings about only the desiredseparation of the monosulfate ester bonding, without attacking theremaining functional groups, mainly the peptide compounds in thesensitive starting material. In the main, the compounds according to thepresent invention are produced in that one treats hirudin-PA in anaqueous, preferably buffered, solution or suspension with an individualaryl sufatase preparation, e.g., of the arylsulfatase of Helix pomatiaat a temperature that is usual for enzymatic processes, such asapproximately 20°-45° C., preferably 25°-30° C. It is preferred that thework be done in a weakly acidic reaction, i.e., at a pH of approximately4-7, in particular from approximately 5-6, which is adjusted with abuffer, such as an approximately 0.03 to approximately 0.3 molarsolution of a salt of an organic carboxylic acid with an alkali metal orwith an organic base, e.g., with sodium acetate or in particular pyridinacetate (of pH approximately 5.4). The ratio of enzyme used to thesubstrate (hirudin) is generally determined by the activity of thepreparation in question, and normally amounts to approximately 1:2 toapproximately 1:100, in particular from approximately 1:5 toapproximately 1:20; it is preferable to use the purest possible enzymepreparations and those that are the most active. Since thearylsulfatases catalyse not only the separation but also theintroduction of the sulfate group and bring about an adjustment of anequilibrium of the starting and the end substances, it is advantageousto establish the optimal concentration, quantity ratios to the substrateand times for desulfatisation for each enzyme preparation, by means ofpreliminary experimentation. As a rule, however, the reaction ends in afew minutes; the quality of the reaction products is not influenced evenby longer contact (up to approximately 4 hours) with active enzyme(e.g., if the reaction mixture is left to stand).

The course of the enzymatic desulfatisation can be monitoredbioanalytically on the samples that have been taken: practically, oneproceeds, for example, in that the enzyme activity is destroyed bybriefly (for approximately 3 minutes) heating the sample toapproximately 100° C. and the substrate is treated with acarboxypeptidase Y. (The carboxypeptidase Y breaks down the peptidechain from the carboxy side, in that the amino acids are split off oneafter the other by splitting the respective amine bonds). Normally, thebreakdown of the peptide chain is so advanced after some 15 minutes thatthe sulfatised and/or free amino acid in position 64 (Tyr64) is splitoff completely and thus accessible to determination in a conventionalamino acid analyser.

The E,D-shortened desulfatohirudins result through splitting off of thetwo C-terminal amino acid building blocks Glu and Asp in the course ofthe hydrolysis of hirudin-PA. The separation of the mixture that resultsduring this can be monitored preparative by HPLC chromatography. Thedesulfatohirudins-PA possess the same biological properties ashirudin-PA.

The compounds according to the present invention can be present in freeform and as salts. Since they contain free amino groups or amidinogroups, they can also be present in the form of acid additive salts. Inparticular, physiologically tolerable salts with normal, therapeuticallyusable acids are suitable as additive salts. The halogen hydracids, forexample, hydrochloric acid, and even sulfuric acid and phosphoric orpyrophosphoric acid can be cited as inorganic acids. As organic acids,one can use sulfonic acids, for example, dibenzol or p-toluol-sulfonicacid or lower alkane sulfonic acids such as methane sulfonic acid, onthe other hand carboxylic acid, such as acetic acid, lactic acid,palmitic acid, and stearic acid, malic acid, tartaric acid, ascorbicacid and/or oxalic acid. Since, on the other hand, the compoundsaccording to the present invention also contain free carboxyl groups,they can be present as the salt of a base, e.g., as sodium, potassium,calcium or magnesium salt, or as ammonia salt or as salt of aphysiologically tolerable organic base that contains nitrogen.

According to the procedure, the compounds according to the presentinvention can be extracted in free form or in the form of acid additivesalts, inner salts, or salts with bases. The free compounds can beextracted from the acid additive salts in the known way. From the latterone can extract therapeutically useful acid additive salts by conversionwith acids, e.g., with such acids as form the above named salts,evaporation or lyophilisation. The inner salts can be extracted byadjustment of the pH to a suitable neutral point.

The present invention also relates to pharmaceutical preparations thatcontain the compounds according to the present invention, or theirtherapeutically useful salts, optionally together with a pharmaceuticalcarrier and/or accessory agents.

These compounds can be used in particular in the presence of the aboveindications if, for example, they are used parenterally (intravenously,intracutaneously, intramuscularly, or subcutaneously), orally, ortopically. In the first instance, the dosage used will depend on thespecific form of use and the purpose of the therapy or prophylaxis. Thesize of the individual doses and the administration regimen can be bestdetermined on the basis of an individual assessment of the particularcase; the methods needed to determine relevant blood factors arefamiliar to the expert. In the normal case, the therapeuticallyeffective quantity of the compounds according to the present invention,administered by injection, is in the dosage range of approximately 0.005to approximately 0.1 mg/kg body weight. The range from approximately0.01 to approximately 0.05 mg/kg body weight is preferred.Administration is by intravenous, intramuscular, or subcutaneousinjection. Accordingly, pharmaceutical preparations for parenteraladministration in single dose form contain approximately 0.4 toapproximately 7.5 mg of the compound per dose, according to theinvention, depending on the method of administration. In addition to theeffective agent, these pharmaceutical preparations normally contain abuffer, e.g., a phosphate buffer that is intended to keep the pH betweenapproximately 3.5 and 7, as well as sodium chloride, mannitol orsorbitol to adjust isotonicity. They can be present in freeze-dried ordissolved form, in which connection the solutions can advantageouslycontain an antibacterial conservation agent, for example, 0.2 to 0.3%4-hydroxybenzoic acid methyl ester or -ethyl ester.

A preparation for topical use can be in the form of an aqueous solution,lotion or jelly, an oily solution or suspension, or a salve thatcontains grease or emulsion. A preparation in the form of an aqueoussolution is obtained, for example, in that one dissolves the substanceaccording to the present invention or a therapeutically useful salttherefrom in an aqueous buffer solution of pH 4 to 6.5 and, if desired,adds an anti-inflammatory agent and/or a polymer adhesive, for examplepolyvinylpyrrolidon, and/or a conserving agent. The concentration of theeffective substance is approximately 0.08 to approximately 1.5 mg,preferably 0.25 to 1.0 mg in approximately 10 ml of a solution or 10 gof a jelly.

An oily applicational form for topical administration is obtained, forexample, by suspending the substance according to the present inventionor a therapeutically useful salt thereof in an oil, with the optionaladdition of a bulking agent such a aluminum stearate and/or surfacereactants (tensides) of which the HLB (Hydrophilic-lipophilic-balance)factor is below 10, such as fatty acid monoesters of polyvalentalcohols, for example, glycerine monostearate, sorbitane monolaurate,sorbitane monostearate or sorbitane monooleate. A greasy salve isobtained, for example, by suspending the substances according to thepresent invention or the salts in a coatable grease base, optionallywith the addition of a tenside having an HLB factor of less than 10. Anemulsion salve is obtained by preparation on a powdered milk sugar baseusing an aqueous solution of the substance according to the presentinvention or the salts in a soft, coatable grease base with the additionof a tenside, the HLB factor of which is less than 10. All these formsfor topical administration can contain preservatives. The concentrationof the effective substance is approximately 0.08 to approximately 1.5mg, preferably 0.25 to 1.0 mg in approximately 10 g of the basic mass.

In addition to the above describe pharmaceutical preparations and theanalogs therof, which are suitable for direct-medical use on the humanbody or on the body of a mammal, the present invention applies topharmaceutical preparations and compounds for medical use outside theliving human or mammal body. Such compounds and preparations are usedprimarily as anticoagulants in blood that is subjected to circulation ortreatment outside the body (e.g., dialysis in artificial kidneys),conservation or modification (haemoseparation). In their compositionpreparations of this kind, such as stock solutions or packaged insingle-dose form, are similar to the above described injectionpreparations; however, more advantageously, the quantities orconcentrations of the substances are related to the volume of blood thatis to be treated or, more precisely, to the thrombin content of suchblood. In this connection, it is to be noted that the substancesaccording to the present invention (in free form)

(a) deactivate an approximate 5-fold quantity by weight of thrombin;

(b) are physiologically harmless even in larger quantities;

(c) are separated out of circulating blood even at high concentrations,so that there is no danger of overdose even in the case of transfusion,for example. According to the specific purpose, the suitable doseamounts to approximately 0.01 to 0.1 mg substance per liter of blood,and even then the upper limit can be greatly exceeded with no danger.

The bioanalytical use of the compounds according to the presentinvention and their salts used to analyze thrombin also form part of theobjects of this invention, as well as preparations that contain thesubstances according to this invention, used for this purpose, e.g.,mixtures of solids and above all solutions, in particular aqueoussolutions; this can advantageously contain inert accessory substances inaddition to the precise quantity or concentration of the substanceaccording to the invention (also in the form of a salt), for example,those discussed above among the injection preparations which fulfill,for example, a stabilising and/or conserving role. These preparationsare used to analyse thrombin in an analogous manner during bioanalysis.

Furthermore, the compounds according to the present invention can beused to preserve blood. To this end, blood preservatives are added tothese compounds in a quantity of 0.1-2%-wt.

In the preceding description and in the claims, the abbreviateddesignations for amino acids and their radicals are used in keeping withthe generally accepted rules of nomenclature and relate to alpha-aminoacids and the radicals thereof, of the naturally occurring L-series.

EXAMPLE 1

2.5 g crude hirudin (extracted by fractionated precipitation withacetone) were dissolved in 20 ml elution buffer and put in a SephadexG-75 medium column (3.8×150 cm). 0.05M triethanolamine, 0.4M NaCl, 0.02%NaN₃, pH 7.8 was used as an elution buffer. Throughput 55 ml/hr;fraction volume: 9.7 ml.

The fractions that were inhibitor active against thrombin were combined(300 to 400 ml), desalinated in an Amicon ultrafiltration cell withUM-05 membrane, and lyophilised.

The lyophilisate was subjected to anion exchange chromatography on DEAEcellulose (DE-52, Whatman; 2.5×100 cm).

Equilibrating buffer: 0.03 M ammonium acetate, pH 6.5.

Throughput: 25 ml/hr; fraction volumes: 8.3 ml.

After dissolution 1.4 g of the inhibitor was added to 10 ml of theequilibrating buffer.

The column was developed with equilibrating buffer until thechymotryptic inhibition activity had been eluated. The elutriation wascarried out with a sodium acetate buffer of pH 6.0 composed as follows:0.2M NaAc, 0.19M NaCl, 0.02% NaN₃.

The thrombin inhibiting fractions were combined, desalinated asdescribed above by ultrafiltration, and lyophilised.

An equilibrating buffer of 0.2M sodium acetate, 0.19M NaCl, pH 6.0, wasused for the subsequent chromatography on DEAE-Sephadex A-25 (column1.9×68 cm). Throughput: 11 ml/hr; fractions volumes: 3.7 ml.

After dissolution and adjustment of the pH value, 6.3 mg of theinhibitor was added to 10 ml of the equilibrating buffer.

The column was developed for 2 hours with equilibrating buffer, thenconverted to the same buffer of pH 5.0, and equilibrated for a further14 hours.

The column was elutriated with a linear pH gradient (the same buffer asabove, pH 3.7).

The thrombin inhibiting fractions, which had been elutriated atapproximately pH 4.6 to 4.7, were concentrated to approximately 3 ml.

Finally, lyophilisation was carried out, desalination completed onSephadex G-25 with water as the washing agent, and lyophilisationcompleted once more.

The lyophilisate was broken down by means of RP-HPLC on Lichrospher 100CH-18/2, 5μ, 4.6×250 mm. Elutriation was carried out with 0.1% trifluoracetic acid in water (V-V; Buffer A) and 0.1% trifluoracetic acid withacetonitrile with 40% buffer A (V-V; Buffer B). The elutriation tookplace isocratically with a solution of 63% buffer A and 37% buffer B(V-V). Flow: 1 ml/minute. Detection: at 214 and 254 nm.

The eluate obtained during a retention time of 9.6 minutes containshirudin-PA, which was obtained after lyophilising in substance.

EXAMPLE 2

Production of the desulfated derivatives by incubation witharylsulfatase (ARS)

ARS-parent solution: 0.1 ml ARS suspension was diluted with 0.1Mammonium acetate, pH 5.5, to 2.5 ml and desalinated on a PD 10 column.

Hirudin-PA parent solution: 2 mg/ml buffer.

Buffer: 0.1M ammonium acetate, ph 5.5

Hirudin-PA parent solution and ARS parent solution were mixed in theproportion of 1:10 (V/V) and incubated at 25° C. The time sequence ofthe reaction was monitored by HPLC. After an incubation period of 24hours it was heated to 90° C. for 3 minutes and the mixture broken downby HPLC under the following conditions:

Column: Lichrospher 10 CH-I8/2, 5μ, 4.6×250 mm.

Buffer A: 0.1% trifluoracetic acid in water (V/V)

Buffer B: 0.1% trifluoracetic acid in acetonitrile with 30% buffer A(V/V)

Flow: 1 ml/minute

Detection: at 214 nm

Temperature: 25° C.

Elution: isocratically with 66% buffer A and 34% buffer B (V/V)

EXAMPLE 3

Production of the reduction product of hirudin-PA

Hirudin-PA solution (2 mg/ml buffer) katepsin C parent solution (20 U/1,14 ml) were mixed in the proportion of 1:5 (V/V) and incubated at 37° C.The course of the proteolysis was monitored by RP-HPLC. The split batch[Spaltansatz] is separated off preparitively by means of HPLC. Theconditions were as cited in Example 2.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A hirudin-PA compound ofthe formula I: ##STR6## in which the above letters representproteinogenic amino acids corresponding to the IUPAC nomenclature, saidamino acids being peptidically bonded, and in which Y^(*) is tyrosin,tyrosin-O-sulfate, or derivatives thereof, shortened by up to 2 aminoacids at the N-terminus, and the amino acid chain at the C-terminus isshortened by the sequence ##STR7## or, if Y^(*) is tyrosin, the aminoacid chain is shortened by the sequence D E or E at the C-terminus,or apharmaceutically useful salt thereof.
 2. Hirudin-PA derivatives as inclaim 1 of the general formula I, wherein the amino acid chain isabbreviated at the N-terminus by the sequence I or IT.
 3. The hirudin-PAcompound as in claim 1, wherein Y^(*) is tyrosin-O-sulfate or apharmaceutically useful salt thereof.
 4. A desulfato hirudin-PA compoundas in claim 1, wherein Y^(*) is tyrosin or a pharmaceutically usefulsalt thereof.
 5. A desulfato hirudin-PA compound as in claim 1 havingthe formula: ##STR8## wherein Y^(*) is tyrosin is a pharmaceuticallyuseful salt thereof.
 6. A desulfato hirudin-PA compound as in claim 1having the formula: ##STR9## wherein Y^(*) is tyrosin or apharmaceutically useful salt thereof.
 7. The hirudin-PA compound ofclaim 1, which comprises 66 amino acids having a molecular weight of7087 and a specific antithrombin activity of 680-720 IU/mg.
 8. A methodfor inhibiting blood clotting in humans or animals, which comprisesadministering a therapeutically effective amount of the hirudin-PAcompound of claim 1 to a subject.
 9. The method according to claim 8,which comprises administering about 0.005 to about 0.1 mg/kg by bodyweight of said compound to the subject by intravenous, intramuscular orsubcutaneous injection.
 10. A composition comprising an effectivethrombin inhibiting amount of the hirudin-PA compound of claim 1 and apharmaceutically acceptable carrier therefor.
 11. A topical preparationcomprising the composition of claim 10.